SHP-1/STAT3訊息傳遞路徑於肝癌標靶治療之分子機制研究

Abstract

中文摘要 肝癌盛行於全世界，並且為臺灣發生率及死亡率最高的腫瘤之一。早期肝癌的治療以外科切除為主，但是大約只有百分之20至30的病患能夠以手術治療，對於晚期肝癌或手術後復發及發生轉移的病患，化學治療為主要的療法。不過，長久以來的化療成果並不顯著。近年來，分子標靶藥物已為癌症治療注入一股新的希望。於2007年，Sorafenib成功成為第一個由美國FDA核准上市的小分子標靶治療藥物。Sorafenib作用於多種分子激酶的功能抑制，以有效控制腫瘤細胞增生。在我們最近的研究顯示，有效的併用 Sorafenib 及其他訊息傳遞抑制藥物可達到增敏作用，以強化抗癌效果。我們發現 Sorafenib 可以有效克服肝癌細胞對於 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)的抗性並顯著地造成細胞凋亡的現象(Chapter 1).。在這項研究中，我們發現 Sorafenib 透過增加SHP-1去磷酸激酶的活性來降低 STAT3 於細胞訊息傳遞路徑中的活化。STAT3 是一個重要的轉錄因子，其下游訊息可活化一系列負責細胞增生及存活的分子。我們已證實有效的抑制 STAT3 活化，可以造成顯著的抗癌效應。我們更進一步討論 Sorafenib 對於 STAT3之去活化反應，是否和它的激酶抑制作用有關。我們設計了一系列小分子 Sorafenib 衍生物，但不具有其原始的激酶抑制作用。在一連串的分子及細胞實驗驗證下，我們發現 Sorafenib 對於 STAT3 的去活化現象和其激酶抑制作用無關。藉由此一系列衍生物最具代表性的小分子 SC-1，我們可以發現相似的癌細胞生長抑制作用。在小鼠肝癌模式中，我們更發現衍生物 SC-1 有優於 Sorafenib 的表現。此一研究為Sorafenib 的應用帶來嶄新的契機(Chapter 2)。除此之外，我們發現另一多激酶抑制標靶藥物，Dovitinib，亦有此一影響SHP-1/STAT3相關訊息傳島之功能，並可造成原先對於Sorafenib具抗藥性之細胞死亡(Chapter 3)。為延續 Sorafenib 相關研究，我們針對Sorafenib如何增加SHP-1活性做更近一步的探討(Chapter 4)。SHP-1的自體抑制(autoinhibition)為一重要決定其催化活性的原因之ㄧ。我們發現Sorafenib不只可直接作用於SHP-1調控其活性以外，並可影響N端SH2 domain與C催化端之聯結，藉以呈現開啟之型態以增加去磷酸激酶活性。去除N-SH2 domain及單一變異D61A的SHP-1，無法呈現Sorafenib原有對於肝癌細胞之分子特性。同時，我們也在臨床肝癌病人檢體中發現，低表現SHP-1及高強度p-STAT3之關連性Sorafenib調控 SHP-1 之去磷酸根活性來達到有效的 STAT3 抑制作用的研究中，對於下一階段的設計及合成 STAT3抑制劑(亦為SHP-1 活性增強劑)將因具有足夠之分子依據而更具體。由衷希望，在不久的將來，我們的研究工作可以提供更精進的治療選擇，並造福更多肝癌患者。

Abstract

Human hepatocellular carcinoma (HCC) is the fifth most prevalent solid tumor in the world and the fourth main inducer of cancer-related death. Surgical resection, the only curative treatment for HCC, is feasible in only 20-30% of the patients at diagnosis. Unfortunately, systemic chemotherapy or liver transplantation is limited for patients with HCC. In 2007, sorafenib (NexavarR), a multiple kinase inhibitor, has shown survival benefits in patients with advanced HCC and become the first clinically approved drug for HCC. First, we have showed that sorafenib sensitizes HCC cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) through the inhibition of signal transducer and activator of transcription 3 (STAT3) (Chapter 1). In this study, we found that sorafenib inhibits STAT3 through up-regulating the activity of Src homology-2 containing protein tyrosine phosphatase-1 (SHP-1), a protein tyrosine phosphatase and a key inhibitory regulator of STAT3, in HCC cells. To examine whether sorafenib’s effect on STAT3 is related to its kinase inhibitory activity, we further generated a series of sorafenib derivatives which lack activities on kinases. Interestingly, we have observed SC-1, a sorafenib derivative which is close to sorafenib structurally but no kinase activity, showed even better in vivo activity than sorafenib in HCC tumors. These results suggest that SHP-1-dependent STAT3 inhibition is a major kinase-independent target of sorafenib (Chapter 2). In addition to sorafenib, we also found that dovitinib, another multiple kinase inhibitor, shows the anti-HCC effect via a SHP-1/STAT-dependent signaling pathway and overcomes the resistance of sorafenib (Chapter 3). Finally, to elucidate the molecular mechanism by which sorafenib increases SHP-1 phosphatase activity, we demonstrated the conformation-based changes in SHP-1 to promote catalytic activity after sorafenib treatment (Chapter 4). Sorafenib increases SHP-1 activity in vitro and in vivo, indicating that sorafenib affects phosphatase activity directly. Based on a series of deletion mutants of SHP-1, we observed that N-terminal SH2 domains (N1) strongly involved in sorafenib-induced STAT3 inhibition and apoptotic effect. Moreover, D61, a critical residue in N1 responsible to form inhibitory salt bridge with catalytic domain, also abrogates the biological effect of sorafenib. In co-IP experiments, sorafenib impairs the interaction between N1 and PTP directly. Furthermore, the role of elevated SHP-1 served as tumor suppressor of HCC was confirmed in cells expressed constitutively activate mutant (dN1 and D61A). Together, we propose a conformational change model for sorafenib-induced SHP-1 activity. Sorafenib potentially opened the inhibited structure of SHP-1 through impairing the linkage between N-SH2 and PTP domains to increase phosphatase activity.